The so-called “Enzyme-linked immunospot assay” (ELISPOT) is used inter alia for measuring the antigen-specific activity of blood cells. The test is usually carried out in microtiter plates having 96 wells, each well having on the bottom a membrane on which a local color reaction around individual activated cells is formed in the event of a positive outcome of the test. The evaluation is carried out by simply counting the points or by using digital image processing systems, in which case the size and intensity of the points can also be quantified in addition to the number of points. The color reaction is based on the detection of locally formed messengers, which are bound via membrane-coupled primary antibodies. These locally bound messengers are then visualized by binding a second antibody, directed against the same messenger, which is directly or indirectly coupled with a color reaction.
Until now, only the number of reactive cells has been measured in the activity measurement of cells, to be able to draw conclusions therefrom concerning their activity. In this regard, however, it has been discovered that it is not so much or not only the number of reacting cells which is important, but rather above all the quality of the reaction of the individual cells. For instance, individual cells may react very differently to the same antigen, which is manifested in the quantity of messengers released and therefore the quantity of color reactions, mentioned in the previous section, of each individual cell. EP 0 957 359 A2 describes an ELISPOT method in which not only the number of reactive cells but also the intensity of the overall reaction is determined. This allows substantially more extensive conclusions concerning the activity of the cells.
One fundamental problem of the ELISPOT method, however, relates to the targeted detection of cells which secrete two or more different messengers in the presence of an antigen. Another problem involves detecting these cells separately from other cells, which secrete only one messenger.
As already mentioned in the introduction, the measurement of cells in the scope of the ELISPOT method is generally based on color reactions which are due to chemoluminescent or fluorescent properties of corresponding dyes. The filter systems conventionally used for chemoluminescence or fluorescence measurement are usually based on a single filter set which, in respect of the excitation and emission of a dye, is transmissive only for light of a particular wavelength range. Admittedly, so-called “dual filters” are by now also used. A disadvantage with this, however, is that these filters are transmissive for a comparatively wide spectral range. Since the dyes can generally absorb and emit light outside their “ideal” spectral range, using dual filters for the measurement of cells which secrete two or more different messengers leads to a significant restriction of the measurement accuracy. This problem is exacerbated by the fact that the messengers are usually secreted in different amounts by the cells.
It could therefore be helpful to provide an ELISPOT method which avoids the disadvantages of the prior art and, in particular, is suitable for a sensitive measurement of cells which secrete two or more different messengers.